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Vol. 19, Issue 5, 2339-2347, May 2008
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Department of Biochemistry and Molecular Biology and the Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic, Rochester, MN 55905
Submitted November 12, 2007;
Revised February 8, 2008;
Accepted March 3, 2008
Monitoring Editor: Josephine Adams
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Paluch et al., 2006). Suprastimulation (SS) of isolated pancreatic acini with high concentrations of secretagogue such as the G protein-coupled receptor agonist cholecystokinin (CCK) or the CCK analogue caerulein (CER) leads to a block in zymogen secretion that is accompanied by significant alterations in cell morphology and the actin cytoskeleton, including cell blebbing (Burnham and Williams, 1982; Adler et al., 1984; O'Konski and Pandol, 1990; Torgerson and McNiven, 1998). In addition, SS induces vacuole formation (Watanabe et al., 1984; Waterford et al., 2005), missorting and premature activation of nascent zymogens (Saito et al., 1987; Husain et al., 2005) as well as aberrant zymogen release from the basal membrane domain (Scheele et al., 1987). Indeed, SS results in cellular responses similar in many aspects to that observed in pancreatitis (Bhatia et al., 2005; Pandol et al., 2007), indicating its relevance as a disease model both in vitro and in animals (Lampel and Kern, 1977).
Along with actin, the actin-based motor protein myosin II undergoes a dramatic relocalization from the apical to the basolateral domain during SS of acini. Indeed, the activation of an actin–myosin network at the cell base seems to result in the formation of a contractile ring that mediates the protrusion of acinar cell blebs. In addition, these membranous extensions are extremely dynamic, forming within minutes of SS and undergoing cytoplasmic streaming as well as amoeboid-like movements. Importantly, cell blebs induced by SS do not seem to represent an apoptotic death response, because they can rapidly recede and reabsorb back into the cell upon removal of the insult (Torgerson and McNiven, 1998). Furthermore, the dramatic SS-induced changes in actin cytoskeletal organization in isolated acini seem to mimic those observed in the intact organ (Fallon et al., 1995; Jungermann et al., 1995). However, the mechanisms regulating changes in actin dynamics following SS are unclear as to whether reorganization of the actin cytoskeleton directly contributes to acinar cell injury or is simply a consequence of cell damage.
The actin-binding protein cortactin is a well-known component of the actin polymerization machinery in many cell types, and it is involved in the formation of branched actin networks that mediate membrane protrusion (Weed and Parsons, 2001; Weaver et al., 2003). In addition, cortactin is phosphorylated downstream of and regulates actin dynamics in response to various signaling pathways. Indeed, cortactin was identified as a major substrate of v-Src in transformed cells, and it is phosphorylated by various members of the Src family of nonreceptor tyrosine kinases (Daly, 2004; Lua and Low, 2005). The Src family kinase members c-Src, Yes, and Lyn have been reported to be expressed in pancreatic acinar cells as well as activated in response to SS (Nozu et al., 2000; Lynch et al., 2004; Pace et al., 2006). Therefore, we hypothesized that the aberrant actin reorganization and cell blebbing in response to SS could be a result of inappropriate activation of Src and subsequent increased cortactin tyrosine phosphorylation. Moreover, because Src inhibitory drugs are commercially available, these could be used to test whether inhibition of Src activity in animal models of pancreatitis alleviates disease severity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Secondary antibodies for immunofluorescence, conjugated to Alexa 488 or Alexa 594, and ProLong Gold were from Invitrogen (Carlsbad, CA). HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies for Western blotting were from Affinity BioReagents (Golden, CO). Collagenase type IV was from Worthington Biochemicals (Lakewood, NJ). PP2 and SU6656 were from Calbiochem (San Diego, CA), and they were dissolved in dimethyl sulfoxide (DMSO) as a 1000x stock. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Preparation and Use of Acini
Acini were harvested in the presence of 100 U/ml collagenase in a buffer containing 20 mM HEPES at pH 7.4, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 10 mM sodium pyruvate, 0.1% bovine serum albumin, and 0.01% soybean trypsin inhibitor as described previously (Torgerson and McNiven, 1998). After isolation, acini were filtered through a 70-µm mesh (Fisher Scientific, Pittsburgh, PA), and they were kept at 37°C for 2 h before use. When indicated, acini were treated during the 2-h incubation with either 20 µM PP2 or 10 µM SU6656. Viability before use was >95%, as indicated by trypan blue exclusion. For experiments involving adenoviral-mediated infection, acini were cultured for 16 h in RPMI 1640 medium (Mediatech, Herndon, VA) with 10% fetal calf serum, 20 mM nicotinamide, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA). Adenovirus was prepared by the University of Iowa Gene Transfer Vector Core by using the RAPAd system (U.S. Patent 6830,920B2, Anderson et al., 2000). The cDNA sequence corresponding to rat cortactin isoform B (GenBank accession no. NM_021868) was used for generating the wild-type (WT) cortactin construct. The cortactin tyrosine phospho-mutant in which tyrosines 384, 429, and 445 were mutated to phenylalanines (M3 cortactin) was subsequently generated using the Stratagene QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Virion encoding for WT cortactin or M3 cortactin were incubated with cells at 107 plaque-forming units/ml. To detect the expression of either WT cortactin or M3 cortactin, 16 h after infection, cells were processed for immunofluorescence or Western blot analysis by using the 4F11 or AB3 antibody, respectively.
Immunofluorescence Staining
Acini were plated on plain glass coverslips, fixed with 2% paraformaldehyde, and permeabilized with 0.6% Triton X-100 as described previously (Torgerson and McNiven, 1998). Acini were then blocked with 5% normal goat serum, incubated with primary antibodies (4F11, diluted 1:1000; SC-18, diluted 1:300; and anti-Yes, diluted 1:1000) for 1 h at room temperature, washed, and incubated with secondary antibodies (Alexa 488- or Alexa 594-conjugated, diluted 1:500) with or without rhodamine-conjugated phalloidin (100 nM; Invitrogen) for 30 min. After washing, acini were mounted in ProLong Gold (Invitrogen, Carlsbad, CA). Acini with six to 12 cells were randomly chosen and imaged using a Zeiss LSM510 confocal microscope (Thornwood, NJ). Images were processed using Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA). For quantitation of blebbing, the number of blebs was counted along with the number of cells. The result, reported as bleb number per 100 cells, represents the mean ± SEM for at least three independent experiments.
Immunoprecipitation and Western Blot Analysis
Acini or tissue were homogenized in a buffer containing 50 mM Tris at pH 7.2, 150 mM NaCl, 0.5 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, 1 mM Na3VO4, 25 mM NaF, 1% NP-40, and Complete (Roche Diagnsotics, Indianapolis, IN) protease inhibitor cocktail. Lysates were either boiled in 1x Laemmli sample buffer before Western blot analysis according to standard procedures or used for immunoprecipitation. In this case, lysates were incubated with 5 µg/ml primary antibody for 2 h at 4°C, followed by addition of 4 mg of protein A beads for 1 h in the same buffer. The beads were then washed three times, boiled in 1x Laemmli sample buffer, and analyzed by Western blot. The following antibodies were used at the indicated dilutions for Western blot analyses: AB3 (1:1,000,000), 4G10 (1:1000), SC-18 (1:500), anti-PY416 (1:1000), anti-Yes (1:5000), H-12 (1:200), anti-Fyn (1:200), and anti-Lyn (1:200). Band intensity was quantified by densitometry using the Bio-Rad Image Analysis System with Molecular Analyst software and a Bio-Rad Model GS-700 imaging densitometer (Bio-Rad, Hercules, CA). The levels of phosphorylated protein were normalized to total levels of the respective protein. For coimmunoprecipitation experiments, the levels of coimmunoprecipitated protein were normalized to the levels of the primary immunoprecipitated protein, i.e., the protein against which the antibody used for immunoprecipitation was made. The reported results are based on quantitation performed using at least two independent experiments for each assay.
Blood and Tissue Preparation
Animals (6 animals in each group) were given single intraperitoneal injections of saline (control) or 20 µg/kg caerulein in saline by using standard procedures. Blood and tissue samples were harvested 6 h later, and either frozen or fixed in 4% formaldehyde. When used, PP2 was dissolved in DMSO and injected intraperitoneally (3 mg/kg in 0.1 ml) 30 min before caerulein. Animals pretreated with vehicle were given DMSO only.
Morphological Examination and Assays
Five-micrometer sections of paraffin-embedded pancreas were stained with hematoxylin and eosin (H&E). For immunohistochemistry of pancreas, 5-µm sections of tissue embedded in Optimal Cutting Temperature compound (Tissue-Tek, Sakura Finetek, Torrance, CA) were fixed with 4% paraformaldehyde and immunostained as described above. Serum amylase activity was measured colorimetrically using the Phadebas assay (Pharmacia Diagnostics, Portage, MI). Edema was measured by subtracting the dry weight from the wet weight, and the data are reported as a percentage of wet weight.
Statistical Analyses
The results reported represent the mean ± SEM of values obtained from three or more experiments, and they were compared using the Student's t test when the data consisted of only two groups or analysis of variance (ANOVA) when comparing three or more groups. If ANOVA indicated a significant difference, the data were analyzed using Tukey's method as a post hoc test for the difference between groups. A p value of 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In contrast, stimulation with supraphysiologic concentrations of CCK (10 nM) induces a marked redistribution of actin from the apical to the basolateral domain where it seems to support the formation of large, dynamic blebs (Figure 1a''; Burnham and Williams, 1982; Adler et al., 1984; O'Konski and Pandol, 1990; Torgerson and McNiven, 1998). Interestingly, we observed that the actin-binding protein cortactin is also localized apically after stimulation with physiological concentrations of CCK (Figure 1b') and that it becomes redistributed in a manner similar to that of actin after SS (Figure 1b'').
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; Daly, 2004; Lua and Low, 2005). Therefore, we tested whether SS leads to tyrosine phosphorylation of cortactin and might thus accompany actin and cortactin redistribution. Here, pancreatic acini were isolated from rats, treated with various concentrations of CCK for 2 min, a time frame in which bleb formation occurs in the presence of SS, lysed, and subjected to immunoprecipitation by using antibodies against cortactin. Subsequently, the immunoprecipitated samples were analyzed by Western blot for tyrosine phosphorylated cortactin by using anti-cortactin and anti-phospho-tyrosine (4G10) antibodies. Indeed, supraphysiological concentrations of CCK stimulated a two- to threefold increase in cortactin tyrosine phosphorylation compared with basal conditions or stimulation with physiological CCK concentrations (Figure 1c). A more detailed analysis of SS-induced cortactin tyrosine phosphorylation indicated that this phosphorylation was rapid, peaking around 2 min, and that it was sustained for >30 min (Figure 1d).
Different Src family kinases (c-Src, Yes, and Lyn) have been found to be activated in response to SS of pancreatic acini (Nozu et al., 2000; Lynch et al., 2004; Pace et al., 2006); therefore, they might mediate cortactin tyrosine phosphorylation. Thus, we first used immunofluorescence analysis to determine the localization of Src (anti-pan-Src antibody) with respect to cortactin in isolated acini under basal conditions and after stimulation with different concentrations of CCK. Resting cells displayed a marked colocalization between Src and cortactin at the apical membrane domain adjacent to the lumen, which extended down along the lateral membrane domains (Figure 2aa'). This codistribution of Src and cortactin was maintained after stimulation of acini with physiological concentrations of CCK (0.1 nM) over 30 min (Figure 2ee'). Interestingly, after just 5 min of SS (10 nM CCK), both Src and cortactin translocated away from the apical lumen (Figure 2bb'), and by 30 min Src they seemed to become largely cytosolic, whereas cortactin accumulated along the necks of newly protruding blebs (Figure 2cc'). Importantly, this dramatic dissociation and redistribution of Src and cortactin was prevented in acini treated with the Src inhibitory drug PP2 (20 µM) for 2 h before SS (Figure 2dd'), supporting that Src-mediated phosphorylation of cortactin leads to its redistribution.
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Activation of Src Induces Acinar Cell Damage after Suprastimulation
As indicated above, several Src family members are expressed in pancreatic acinar cells, and they become activated in response to SS (Nozu et al., 2000; Lynch et al., 2004; Pace et al., 2006). The above-mentioned studies were performed using an anti-pan-Src antibody, which recognizes multiple Src kinase family members. Therefore, to discern the specific Src family member associated with cortactin, lysates from pancreatic acini were subjected to immunoprecipitation by using either the anti-pan-Src or an anti-cortactin antibody, and the samples were analyzed by Western blot for c-Src, Yes, Lyn, and Fyn. Interestingly, although all four Src family members were detected in whole cell lysates, only Yes was immunoprecipitated by the anti-pan-Src antibody; furthermore, Yes coimmunoprecipitated with cortactin (Supplemental Figure S1, a and b). Subsequent analysis of lysates from resting and CCK-stimulated (10 pM–10 nM) acini immunoprecipitated for Yes indicated that Yes is activated after SS, as determined by using the anti-PY416 antibody (Supplemental Figure S1c; Lynch et al., 2004). Finally, immunofluorescence analysis of resting acini and acini stimulated with either physiological (0.1 nM) or supraphysiological (10 nM) concentrations of CCK for 30 min supported that Yes and cortactin colocalize under physiological conditions (Supplemental Figure S1, d and e'), whereas they become redistributed after SS (Supplemental Figure S1, f and f'). Importantly, this relocalization of Yes and cortactin was prevented in acini treated with PP2 before SS (Supplemental Figure S1, g and g'). These results suggest that Yes associates with cortactin under physiological conditions and mediates cortactin tyrosine phosphorylation after SS; however, we cannot rule out a role for other Src family members. Thus, the term "Src" is used from here on with the intended meaning of Yes as well as potentially other Src kinases.
Pharmacological inhibition of Src activity with PP2, a small-molecule inhibitor of multiple Src family kinases, as well as SU6656 (data not shown; see below), another Src family kinase inhibitory drug, prevented the dissociation of the Src–cortactin complex as well as redistribution of cortactin to the necks of basal membrane blebs normally observed after SS (Figures 1 and 2, and Supplemental Figure S1). Therefore, we next tested whether treatment of acini with either of these Src inhibitors might inhibit acinar cell blebbing. Living and fixed acini were imaged after SS with 10 nM CCK both in the presence and absence of treatment with Src inhibitors (PP2, 20 µM; SU6656, 10 µM), and bleb formation was quantified (Figure 3, a–g). In the absence of Src inhibitors, acini were observed to undergo significant blebbing within 30 min of SS (Figure 3, a–a''). In addition, imaging of fixed acini treated in a similar manner and stained for cortactin and actin indicated a relocalization of these proteins to the necks of membrane blebs, as expected (Figure 3, bb'). In striking contrast, acini treated with either PP2 (Figure 3, c–c'') or SU6656 (Figure 3, e–e'') before and during SS with CCK maintained a near normal shape over the 30-min time period. Accordingly, immunofluorescence analysis of Src inhibitor-treated acini revealed that the apical localization of cortactin and actin was maintained (Figure 3d, d', f, and f'). Quantitation of >100 acinar cells indicated a significant reduction (>75%) in blebbing in cells treated with either of the Src inhibitory drugs in combination with SS compared with cells treated with supraphysiological concentrations of CCK alone (Figure 3g). Importantly, Western blot analysis of acini treated with 10 nM CCK (i.e., SS) in the presence or absence of either PP2 (Figure 3h) or SU6656 (Figure 3i) by using the anti-phospho-tyrosine antibody 4G10 indicated that both drugs inhibited cortactin tyrosine phosphorylation. Thus, Src-mediated phosphorylation of cortactin seems to regulate cell blebbing in response to SS of pancreatic acinar cells. Importantly, PP2 did not affect the secretory response of acini (amylase activity) under control conditions (5.6 + 2.3 vs. 6.0 + 2.5%), or in response to physiological (0.1 nM; 18.4 + 1.8% vs. 18.0 + 2.3%) or supraphysiological (10 nM; 11.0 + 1.9% vs. 11.0 + 2.5%) concentrations of CCK.
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) were mutated to phenylalanine and expressed in cultured acini after adenovirus-mediated infection. Subsequently, the effects of this phospho-mutant protein, referred to as M3 cortactin, on cortactin localization and cell blebbing in response to SS were analyzed. Remarkably, acini expressing M3 cortactin maintained an apical cortactin and actin localization after SS; more importantly, they did not undergo cell blebbing (Figure 4, a–a''), which was in marked contrast to adjacent uninfected acini or acini infected with WT cortactin (data not shown). In support of these observations, quantitation of >100 acinar cells indicated a >50% reduction in SS-induced cell blebbing in cells expressing M3 cortactin compared with uninfected control cells or cells expressing WT cortactin (Figure 4b).
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Inhibition of Src Family Kinases Mitigates Disease Severity in the Caerulein Rat Model of Pancreatitis
Stimulation of isolated pancreatic acini or infusion of animals with supraphysiological concentrations of secretagogue represent models for studying pancreatitis (Bhatia et al., 2005; Pandol et al., 2007). Thus, we next tested whether our in vitro results might translate to the intact pancreas in an animal model of pancreatitis. For these studies, we first analyzed Src activation and cortactin phosphorylation over a 30-min time course by using lysates of pancreata harvested from individual rats for each time point after intraperitoneal injection of the CCK analogue CER (20 µg/kg). Similar to our in vitro results (Figure 2g), there was a rapid 2.2-fold activation of Src within 1 min of in vivo SS, and this was sustained over 30 min (Figure 5ac). In addition, Western blot analysis using the anti-phospho-tyrosine antibody 4G10 after immunoprecipitation of cortactin indicated that cortactin tyrosine phosphorylation was increased within 5 min and that it was even more prominent at 30 min (Figure 5b). Remarkably, intraperitoneal administration of 3 mg/kg PP2 30 min before a 30-min stimulation with CER inhibited the SS-induced increase in cortactin tyrosine phosphorylation (Figure 5d). In agreement with these biochemical results, morphological analysis of pancreas sections from control rats and rats treated with CER for 30 min in the presence or absence of PP2 showed that PP2 treatment prevented the CER-induced relocalization of actin and cortactin from the apical to the basolateral domain in vivo (Figure 5, e–g'). Thus, in isolated acini as well as the intact pancreas, inhibition of Src family kinases attenuates Src activation, cortactin tyrosine phosphorylation, and actin and cortactin redistribution in response to SS.
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; Pandol et al., 2007). For these experiments, control animals and rats exposed to 20 µg/kg CER for 6 h in the presence or absence of PP2 were analyzed. An increase in serum amylase levels can be used as an indicator of loss of acinar cell polarity and subsequent cell death, resulting in aberrant release of zymogens into the blood space. In addition, as aberrant zymogen release, cell death, and inflammation increase, the pancreas becomes edematous. Indeed, treatment of rats with high concentrations of CER increased both of these parameters compared with control animals. Importantly, prophylactic treatment of rats with 3 mg/kg PP2 before CER administration markedly reduced the CER-induced aberrant release of amylase (>50% reduction) as well as increase in edema (Figure 6ab). However, most impressive was the morphological improvement of acinar structure as assessed by histology. Sections of normal pancreas displayed a polarized, well-organized arrangement of acini with zymogen granules clustered at the apical lumen (Figure 6c). In contrast, SS with CER resulted in the formation of large intracellular vacuoles and extensive interstitial edema and inflammation (Figure 6d). Remarkably, the pancreata from rats pretreated with PP2 seemed undamaged, with well-organized acini that mimicked control tissue (Figure 6e). To test for an effect of the src inhibitors on inflammatory cell infiltration, we quantified the number of white blood cells observed in 25 high power fields (HPF) of each variable of pancreatic sections from CER-treated mice pretreated with either PP2 or vehicle. We observed a reduction of white cells in pancreatic tissue from the PP2-treated animals (17.0 + 2.0 HPF in the caerulein pancreatitis group to 4.9 + 1.1 [p < 0.005] in the PP2-pretreated group). Vehicle had no effect on this (19.6 + 3.7 inflammatory cells/HPF; p = 0.53). This observation suggests that the inhibition of src activity could also exert some positive protective effects on the pancreas by reducing acinar-based blebbing and inflammation as well.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Adler et al., 1984; O'Konski and Pandol, 1990, 1993; Fallon et al., 1995; Jungermann et al., 1995; Torgerson and McNiven, 1998). The mechanisms regulating this aberrant redistribution are unclear; moreover, it has yet to be determined whether this alteration in actin cytoskeletal organization plays a causal role in acinar cell injury or whether it is simply a result of cell damage. Here, we report an essential role for Src-regulated actin reorganization during acinar cell injury. Furthermore, we demonstrate that the actin-binding protein cortactin, a known Src substrate, mediates this aberrant reorganization of the actin cytoskeleton in a tyrosine phosphorylation-dependent manner. Importantly, inhibition of Src family kinases using either of the small-molecule inhibitors PP2 or SU6656 reduced acinar cell damage both in vitro and in the caerulein rat model of pancreatitis, indicating the potential usefulness of Src inhibitory drugs in the therapeutic treatment of pancreatitis.
Src family kinases, including Yes, have previously been found to be activated after SS of isolated acini (Nozu et al., 2000; Lynch et al., 2004; Pace et al., 2006), and they are implicated indirectly in disassembly of the actin cytoskeleton (Leser et al., 2000; Lynch et al., 2004). Here, we demonstrate a direct role for a SS-induced increase in tyrosine phosphorylation of cortactin by a Src family kinase, most likely Yes, in relocalization of the actin cytoskeleton and acinar cell damage. Treatment of isolated acini with Src inhibitors (PP2 or SU6656) before SS prevented the aberrant redistribution of the actin cytoskeleton and cell blebbing (Figure 3), suggesting that Src activation and subsequent actin reorganization play a causal role in cell damage. However, various downstream Src substrates such as the focal adhesion protein Pyk2 (Lynch et al., 2004), the cell–cell junction protein p120 catenin (Leser et al., 2000) as well as others might be responsible for mediating the altered actin organization and cell damage. Our observation that expression of a cortactin tyrosine phospho-mutant (M3 cortactin) prevents disassembly of the apical network of filamentous actin, while also leading to a significant reduction in cell blebbing after SS (Figure 4), provides a direct link between Src activation and aberrant actin reorganization during acinar cell injury. Most importantly, these findings support the concept that changes in actin cytoskeletal organization are a primary event in acinar cell damage. In essence, no cortactin tyrosine phosphorylation, no aberrant SS-induced actin rearrangement, no cell damage.
In addition to our observations in isolated acini, these findings translated to the caerulein rat model of pancreatitis. Activation of Yes and cortactin tyrosine phosphorylation were rapidly increased in the intact pancreas of stimulated animals; moreover, cortactin and actin underwent a dramatic basolateral redistribution (Figure 5). Importantly, these effects as well as overall disease severity, as indicated by a decrease in serum amylase levels and pancreatic edema, and most remarkably, restoration of tissue integrity to near that of control animals, were alleviated by treatment of animals with the Src inhibitor PP2 before CER administration (Figures 5 and 6). Thus, these results provide an exciting and necessary extension of previous studies implicating Src family kinases in pancreatic acinar cell injury, while highlighting the in vivo relevance of aberrant Src-mediated cortactin phosphorylation and subsequent actin reorganization in cell damage during pancreatitis.
The formation of basolateral cell blebs in isolated acini after SS is thought to be mediated by activation of an actin–myosin contractile network at the cell base (Burnham and Williams, 1982; Adler et al., 1984; O'Konski and Pandol, 1990; Torgerson and McNiven, 1998). Because inhibition of Src activation and cortactin phosphorylation prevented cell blebbing (Figures 3 and 4), potentially the improper Src-induced, cortactin-mediated assembly of actin at the acinar cell base provides a means for actin–myosin contraction at a site in which it is not needed or preferred. We (Torgerson and McNiven, 1998) and others (Burnham et al., 1988) have observed an increase in phosphorylated myosin light chain after SS of acini, and this correlates with activation of myosin II at the necks of cell blebs (Torgerson and McNiven, 1998). Interestingly, tyrosine phosphorylated cortactin has been demonstrated to bind myosin light chain kinase from endothelial cells and increase its activity, as indicated by increased myosin light chain phosphorylation (Garcia et al., 1999; Dudek et al., 2004). Similar to actin and myosin II, we observed that cortactin is redistributed to the necks of cell blebs after SS (Figures 1 and 3). Thus, SS-induced tyrosine phosphorylation of cortactin by Yes may mediate redistribution as well as activation of the actin–myosin contractile network, thereby directly contributing to acinar cell blebbing and injury.
Cortactin is involved in a variety of cellular processes, including migration, adhesion, and vesicle trafficking. In addition, phosphorylation plays an important role in regulating the effects of cortactin on actin dynamics and cytoskeletal organization (Weed and Parsons, 2001; Daly, 2004; Lua and Low, 2005). Thus, the increase in cortactin phosphorylation after SS could lead to undesirable changes in cell–cell contacts, actin–myosin contractility, and/or membrane protrusion, thereby contributing to bleb formation and cell damage. The findings reported here emphasize the importance of understanding how actin dynamics are regulated in pancreatic acinar cells, while providing significant insight into the molecular basis of acinar cell injury. Most excitingly, these results highlight Src as an attractive therapeutic target for the treatment of acute pancreatitis, a disease for which no treatment currently exists.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Mark A. McNiven (mcniven.mark{at}mayo.edu)
Abbreviations used: CCK, cholecystokinin; CER, caerulein; M3 cortactin, a cortactin tyrosine phospho-mutant in which three key tyrosine residues have been mutated to phenylalanine; SS, suprastimulation; WT, wild type.
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